DESCRIPTION (from applicant's abstract) Neuronal growth cones navigate by the precise execution of specific behaviors at a variety of decision points. Although a wealth of literature demonstrates that such growth cone events rely upon dynamic activities of both cytoskeleton and membrane constituents, our knowledge of underlying mechanisms is incomplete. Though cytoskeletal dynamics are being intensely studied, it has been difficult to directly investigate comparable activities of the plasma membrane. The last five years have seen major progress in our understanding of membrane dynamics in systems other than growth cones in large part due to the synthesis and implementation of the fluorescent dye FM1-43. This dye allows real time visualization of membrane events in living cells. The present proposal makes use of this powerful tool to test the overall hypothesis that endocytosis is a highly regulated process that is involved in multiple growth cone activities. Key publications have demonstrated that there are diverse, dynamic membrane stores within the growth cone and that the process of endocytosis is required for outgrowth. Pilot experiments with FM1-43 indicate that endocytosis is a surprisingly active process in neuronal growth cones. The first Specific Aim will test the hypothesis that endocytosis is activity dependent in the growth cone, and that electrical activity can regulate both endocytosis and the fate of endocytotic vesicles. The second Specific Aim will use defined guidance cues to evoke specific growth cone behaviors (such as turning, pausing, and stopping) in order to test the complementary hypothesis that endocytosis in growth cones is more specifically related to pathfinding activity. The third Specific Aim will test the hypothesis that the proximate mechanism controlling growth cone endocytosis and the fate of endocytotic vesicles involves changes in intracellular free calcium. The proposed studies will investigate endocytosis in growth cones with temporal and spatial resolution not previously possible and thus could well advance our understanding of membrane dynamics to the level already achieved for the cytoskeleton.